Figure 5

PF-382 cell line DNA sequencing analysis. (A) TP53 gene sequencing at passage 1. Two mutations in codon 273 are visible (nucleotide 817C > T as originally described, and surprisingly, 818 G > A). The first mutation represents heterozygous status, equal amounts of mutated and wild type allele are detected. The sequencing product shows that the wild-type nucleotide is visible as a strong band and mutated nucleotide is visible as a very weak band in the second mutation. (B) Cloning of PF-382 primary culture and TP53 sequencing after the following months of cell culturing shows that intensity of mutated band has changed. (C) N-RAS DNA sequencing. In order to confirm PF-382 cell line identity, mutation of N-RAS gene was verified. N-RAS sequencing shows a heterozygous mutation in codon 12 (N-RAS, GGT > AGT, Gly > Ser). Mutations are marked with arrows. N - normal, control template; A - colony.